To identify the primary defect in the spe-11 mutant embryos, we have undertaken a detailed phenotypic analysis. Given that the spe-11 terminal phenotype is a round, fragile 1-cell embryo, we have examined two diagnostics of eggshell integrity. First, we have shown that spe-11(hc90) mutant embryos are osmotically sensitive, indicating a disruption in the inner layer of the eggshell, which confers the osmotic barrier. Second, the chitin layer of the eggshell is defective in these mutants. Chitin is observed only in a restricted crescent at the surface of the embryo in the null spe-11(hc90) embryos, in contrast to wild type embryos where chitin is present around the periphery of the embryo. As an additional marker of early embryogenesis, we have investigated the trafficking of intracellular vesicles called cortical granules, which undergo a characteristic translocation during egg activation. The spe-11 mutants are not compromised in the cell cycle dependent process of cortical granule movement because CAV-1::GFP, a marker of cortical granules, exhibits normal trafficking in spe-11 mutants. We also asked if the localization of other egg activation genes was normal in the absence of SPE-11. EGG-3::GFP is normally localized following fertilization in spe-11(hc90) mutants. Thus, the earliest defects we have detected in spe-11 mutants are in eggshell formation. We are continuing this type of analysis using additional cortical and eggshell markers fused to GFP or mCherry. This analysis should help determine which aspects of early development are perturbed by the lack of SPE-11 and which function independently of SPE-11. In the past, we performed a non-complementation screen in order to recover a strong temperature-sensitive allele of spe-11 that could be used for a genetic suppressor screen. Thus far, we have recovered two new alleles of spe-11, spe-11(av33 and av34). Both were found to be a non-conditional alleles that produce truncated protein products, like most of the existing spe-11 alleles. These new alleles behave like the reference allele and thus we believe they too are null alleles. We are continuing this screen in hopes of identifying a temperature-sensitive allele of spe-11 that can be used in a suppressor screen. Alternatively, we hope to at least identify a missense allele, which would also be a good candidate for a suppressor screen. Because this screen has suggested the existence of non-allelic non-complementing alleles, we are planning to isolate these enhancer alleles in the future as they may be quite informative as to the genetic pathways in which spe-11 functions. We have also initiated a genetic suppressor screen using a truncation allele of spe-11, spe-11(bn65). This allele of spe-11 encodes 80% of the full protein length. We anticipate that this screen will isolate dominant and recessive bypass suppressors of spe-11, perhaps that will make the oocyte more sensitive to activation or that facilitate SPE-11 localization or function. We have also generated a transgenic line that expresses GFP:SPE-11 under the endogenous spe-11 promoter and 3UTR. This line expresses GFP:SPE-11 very brightly in the mature sperm and will be used to perform microscopic studies to determine the fate of the SPE-11 protein following fertilization. In addition, we are constructing transgenic animals expressing fluorescent (GFP) in vivo markers of the eggshell using cbd-1 and cpg-1 translational fusions. These transgenics will provide important tools for evaluating the subtle alterations of the eggshell, which will be of great utility in our investigations of the role of SPE-11 in eggshell formation. In addition to the above strategies, we are also performing yeast two hybrid screens to find SPE-11 interactors and are performing structure-function assays on spe-11 variants by asking which variants are capable of rescuing a null.